WO2004062570A2 - Appareil et methode de mesure de la contractilite des plaquettes - Google Patents

Appareil et methode de mesure de la contractilite des plaquettes Download PDF

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WO2004062570A2
WO2004062570A2 PCT/US2003/024052 US0324052W WO2004062570A2 WO 2004062570 A2 WO2004062570 A2 WO 2004062570A2 US 0324052 W US0324052 W US 0324052W WO 2004062570 A2 WO2004062570 A2 WO 2004062570A2
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chamber
opening
valve
flexible
pump
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PCT/US2003/024052
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English (en)
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WO2004062570A3 (fr
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Enrique Saldivar
Jennifer Orje
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Enrique Saldivar
Jennifer Orje
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Priority to AU2003257983A priority Critical patent/AU2003257983A1/en
Priority to EP03815178A priority patent/EP1618373A2/fr
Publication of WO2004062570A2 publication Critical patent/WO2004062570A2/fr
Publication of WO2004062570A3 publication Critical patent/WO2004062570A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4905—Determining clotting time of blood
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing

Definitions

  • the present invention generally concerns blood clotting components and mechanisms.
  • the present invention particularly concerns new devices and methods for studying clotting mechanisms and factors. More specifically, an apparatus and method for measuring and monitoring health and activity of platelets and other clotting factors are described. Most specifically, a clot retractometer and its method of use are provided to measure clot contractility forces as a means to provide a single point "funnel detection" procedure useful in aiding physiological and clinical research and patient diagnosis and monitoring of many diseases, as well as screening populations.
  • a clot form As a result of injury to tissue, such as, for example, a scrape, puncture or a bleeding nose.
  • tissue such as, for example, a scrape, puncture or a bleeding nose.
  • the formation of a clot is a complex, cascading process that is still not completely elucidated, either physiologically or clinically.
  • the clotting phenomenon, or lack thereof, is manifest in numerous clinical conditions, and is relevant to their prognosis.
  • the haemostatic mechanism In response to soft tissue injury, the haemostatic mechanism is activated to stop bleeding and restore vascular integrity. Blood protein and cellular interactions lead to the formation of a platelet plug and ultimately generation of clot comprising platelets and protein fibers. These reactions have to occur rapidly because the amount of blood lost is dependent on the time required to arrest the bleeding. Although rapid stoppage of blood loss is critical in some cases, inappropriate induction of clotting can have devastating effects such as decreased blood flow to the organs and resultant ischemic damage, such as heart attacks and stroke if the clot is not solubilised. To counterbalance these potentially severe consequences the haemostatic system is uses icertain clotting inhibitors, and clot- dissolving enzymes.
  • platelets Following vascular damage, the exposure of flowing platelets to the subendothelial lining allows the establishment of adhesive interactions with the immobilized surfaces. Platelets then become activated due to contact with thrombogenic substrates and stimulation by locally released or generated agonists. Subsequent platelet deposition relies on the binding of plasma-soluble adhesive molecules, and on the externalization of adhesive molecules from the platelets' granular reservoirs, this process conditions the newly recruited monolayer of platelets to become the reactive surface for continuing platelet accrual.
  • thrombin a serine protease
  • fibrinopeptides A and B from the aminoterminal ends of the A ⁇ and B ⁇ chains of the fibrinogen molecule. Cleavage of fibrinopeptide-A is sufficient to initiate clot assembly (4).
  • the monomer units formed initiate a self-assembly process of forming protofibrils. Weak lateral interactions between protofibers increase as the protofibers lengthen, resulting in their alignment and coalescence, to ultimately yield fibers. This process leads to the formation of a network composed of fibrin polymers and spaces filled with fluid. Once the fibrin network is formed, the platelets begin to contract, resulting in a pull on the strands of the fibrin network. Platelet contraction requires active restructuring of the platelet cytoskeleton.
  • Dynamic rearrangements in the cytoskeleton are crucial during platelet activation in both, initial platelet adhesion to surfaces (see figure 1) and platelet to platelet cohesion. Actin polymerization in non-stimulated platelets is limited by monomer-sequestering proteins such as thymosin ⁇ 4, profilin and barbed end- capping proteins such as gelsolin (5-7). Under these conditions, around 2,000 actin filaments are distributed in the cytoskeleton and in the membrane skeleton right under the inner surface of the plasma membrane (8).
  • Rho family - such as cdc42Hs, Rac, and Rho - have been implicated in the formation of filopodia, lamellipodia and focal adhesion plaques in many cell types (12), and the same may occur in platelets.
  • Pathologic alterations of the system may induce a risk of hemorrhage or increase the potential for thrombosis.
  • An example of the former would be the bleeding disorder of hemophilia, which results from a low activity of Factor VIII, a blood clotting protein.
  • An example of the latter would be recurrent venous thrombosis in individuals who have decreased levels of the coagulation inhibitor antithrombin UI. Patients with decreased ability to remove clots, decreased fibrinolytic potential, are also at risk for thrombosis
  • platelets are downregulated by the anti-thrombotic potential of normal endothelial cells, in vivo, and by substances produced by the activated platelets.
  • the majority of pathways that result in inhibition of platelet aggregation and procoagulant activities act by increasing the internal level of cyclic AMP, which activates the cyclic AMP-dependent protein-kinase. This leads to serine-threonine phosphorilation of an array of substrates.
  • clot retraction is seen as a dramatic reduction in clot volume in a process that expels the fluid trapped inside the clot.
  • physiological role of clot retraction is not completely understood, it is assumed that it helps in approximating the edges of a tissue defect and in concentrating the clot in the area of injury (4).
  • One issue that it is clear in clot retraction is that in order for this process to occur normally all haemostatic mechanisms must act in synchrony.
  • the two primary requirements for proper clot retraction to occur are the formation of an appropriate fibrin network and healthy platelets, capable of contracting and anchoring the fibrin network.
  • the structure and formation of the fibrin network are sensitive to pH, ionic strength, calcium concentration, plasma proteins, platelet release products, leukocyte materials, etc. (4).
  • pathological conditions that affect the structure of the fibrin network are diabetes mellitus and multiple myeloma (4; 13). Healthy platelets need to express the integrin ⁇ b ⁇ 3 on their surface to properly anchor the fibrin strands and they need to be metabolically fit for the task. Examples of pathological conditions that affect platelet metabolism are diabetes mellitus and uremia (14). Also, the age of platelets affects their performance, this aspect is particularly important for transfusion purposes.
  • a common method utilized to evaluate clot retraction is quantitation of the fluid volume expelled by the clot during retraction, and estimation of the volume of the residual clot (16). This is a qualitative essay that does not provide information about the force generated during clot retraction.
  • Another known method involves the formation of cylinders or strips of clots, which are then immobilized on one end and anchored to a force transducer (17) on the other. This technique requires mechanical manipulation of the sample and bathing of the clots in a foreign substance that may alter the natural process of clot 5 retraction.
  • Yet another technique utilizes a rheometer to measure the normal force development during cloning and retraction (18).
  • An important limitation of this technique is the high cost of the equipment.
  • Carr's apparatus consists of a cup in which the fluid sample (before clotting) is placed. The opening of the cup is covered by an upper plate, which is coupled to a steel arm attached to a force transducer. As the clot retracts, the force generated is transmitted to the force transducer, where it is measured (1;4;13;14) . Although 15 ' this is a very reliable method, the cost per measurement is high, because this method allows only the measurement of one sample at a time. Also, this equipment has the added complication of the high precision required for its alignment and setup.
  • the primary object of this invention is to provide a novel platelet retractometer that will measure the force developed by platelets during blood clot retraction.
  • Another object in accordance with the present invention is a device that can automatically measure the force developed by platelets during clot retraction, is easy and inexpensive to operate, and provides no exposure of the operator to biohazardous materials.
  • a further, most preferred object is to provide a method for measuring the force developed by platelets during clot retraction, as well as its clinical applications, in both research and patient monitoring.
  • the clinical applications comprise all conditions in which platelet viability and platelet metabolism are impaired, including, but not limited to, diabetes mellitus, chemotherapy, and monitoring of platelet aging for blood transfusion.
  • the device will increase the scope of study in platelet biology by bringing a user- friendly ready-to-use method useful to dissect the mechanisms involved in platelet contraction in both, physiological and pathological conditions.
  • this invention contemplates an apparatus for measuring blood platelet contractility, hereinafter called a "retractometer.”
  • the retractometer has a spherical rigid chamber with an opening in its dorsal aspect. Found inside this chamber is a smaller, spherical, flexible membrane chamber concentrically aligned and isolated from the larger rigid chamber, creating a void space between the walls of the rigid and flexible chambers.
  • the flexible membrane chamber also has an opening in its upper aspect, smaller than and coaxial to the opening in the rigid chamber.
  • the membrane chamber can be manufactured from latex by dipping a mold and withdrawing a thin spherical bag with an opening created by a shaft attached to a spherical mold.
  • the flexible membrane can be latex or any other suitable material.
  • this invention contemplates an alternative retractometer having similar spherical rigid and flexible chambers, and openings in their upper aspect isolating the two chambers from each other and creating a void space between their walls.
  • the variation from the abovedescribed setup is that the tubular chamber leading out of the flexible chamber concentrically and in perpendicular axis through the opening in the rigid chamber, has both ends sealed. This creates a void space that is isolated from the void space of the flexible inner chamber. Through this tubular chamber, runs a glass capillary tubing, coaxial to and longer than the tubular chamber, passing through both ends of the sealed tubular chamber. This creates a continuous passage from outside of the apparatus to the void space of the inner flexible chamber.
  • the distal opening of the capillary tubing is plugged before directly reading the force applied by the retracting clot as the height of the fluid column inside the capillary.
  • This plug can either be a removable type, like a cap or stopper, or a sealed type that is opened by breaking the capillary at an etched or scored point above the sealed tube.
  • a more specific and preferred embodiment of this invention is an automated system for measuring blood platelet contractility of a plurality of samples having an array of retractometer units with valves as described hereinabove.
  • Each unit retractometer is a separate apparatus for measuring blood platelet contractility of a single sample. As described above, it comprises a spherical rigid chamber having an opening in its upper aspect, a smaller, spherical, flexible membrane chamber placed concentrically within the rigid chamber, creating a void space between the walls of the rigid and flexible chambers, and having an opening in its upper aspect that is smaller than and coaxial to the opening in the rigid chamber.
  • a first, attached contiguous tubular passage leads out of the flexible chamber concentrically and in perpendicular axis through the opening in the rigid chamber, creating a void space that is isolated from the void space of the flexible inner chamber.
  • a two-way valve is attached to the distal end of the tubular passage, which in turn, is connected to a second tubular passage. The end distal to the valve is connected to a common pressure transducer.
  • the valves are operated automatically by solenoids, energized and regulated by an electronic circuit. This circuitry is programmed to choose and operate the solenoid valves in a predetermined order written in software by the inventors.
  • An equally preferred embodiment in accordance with this invention is an electronic solenoid valve controller to fully automate a system comprising a number of retractometers and save costs in labor.
  • This embodiment is a system apparatus for automatically measuring platelet contractility in a plurality of samples.
  • the system has a pump mechanically connected to a pump motor, which in turn is electronically connected to a microprocessor having a plurality of pins. One pin is used to turn the pump motor on; a second pin to move fluid in the pump in one direction; a third pin to move fluid in the pump in an opposite direction; and at least one of the remainder of the pins to activate each of an array of solenoid valves.
  • the system also has a voltage divider used to establish the position of the fluid in the pump.
  • a fluid conduit connecting the pump to a hydraulic system having a manifold that connects the pump to each of a plurality of retractometers controlled by solenoid valves.
  • Each retractometer communicates with one of the solenoid valves.
  • a pressure transducer reads each pressure and sends the reading to an analog to digital (A/D) converter connected electronically to the tranducer, the pump motor, the microprocessor, and a computer.
  • A/D analog to digital
  • sequence of events is as follows.
  • a readout position voltage from the voltage divider is entered through the (A/D) converter to the microprocessor, which determines the direction of flow in the pump and activates the pump to adjust the fluid pressure within the system.
  • the pressure is then measured by the pressure transducer connected electronically to the A/D converter, and a target pressure is registered in the microprocessor memory, which is subsequently recorded and displayed by the computer.
  • This apparatus embodiment may have a pump that moves fluid with a sliding piston.
  • the pump is a syringe controlled by a step motor with very fine gradations.
  • the apparatus has an array of eight solenoid valves, each valve communicating with one of eight retractometers. More preferably, the system apparatus is expandable by addition of retractometers and valves. Most preferably, the retractometers are packaged in a cartridge, such that one cartridge is removed after sampling and replaced with another having additional samples, and so on.
  • the apparatus is protected by a protection valve located at the entrance to the pressure transducer to prevent damage to the system and a second protection valve to control access to a fluid reservoir.
  • the subroutines in the analysis program are burnt into the microprocessor.
  • a most preferred embodiment in accordance with this invention is a method for measuring blood platelet contractility.
  • the method comprises the steps of preparing a retractometer according to this invention by applying adhesive to the surface of the inner flexible membrane to avoid slippage of clots.
  • the adhesive can be any suitable substance, for example, collagen Type I suspension.
  • the coated flexible membrane is then pressure conditioned by mounting it on a rubber stopper pierced by a hypodermic type needle attached to a two-way valve. A syringe is attached to one opening of the valve and a second needle is attached to a second opening of the valve, making certain that the reach of the two needles is identical.
  • the membrane chamber is slightly pressurized, the valve to the syringe is closed, communication is opened to ambient fluid. In this manner, the inner and ambient pressures are allowed to equilibrate by siphoning. The fluid level inside the capillary is adjusted to "zero pressure" level.
  • the second step involves loading of the sample into the void created between the two chambers, surrounding and in contact with the outside surface of the flexible membrane chamber. A small amount of oil is added over the sample to avoid drying out. The sample is then allowed, or induced, to clot and the force of the clot retraction is measured in a pressure transducer and recorded.
  • a first step requires calibration of the apparatus above. This entails the microprocessor reading all initial pressures in all retractometers sequentially by opening each solenoid valve, opening the protection valve, measuring the voltage in the pressure transducer and storing the measured value in the temporary memory of the microprocessor. This process is repeated until all the initial pressure values are registered as target values for each of the retractometers.
  • the second step adjusts the value of the hydraulics by opening the protection valve only and activating the pump until the target value is reached. The sample is then loaded into the retractometers, and clot formation is induced.
  • the third step requires opening of the sample valve, measuring the pressure, and closing the sample valve, in that sequence.
  • the measured values are then sent to a text file in a computer, and the new measured value for each retractometer becomes the next target value.
  • This third step is repeated until all samples are measured. The entire process of measuring the clotted samples takes less than one minute.
  • the methods described here are useful in determining platelet activity.
  • the ability to determine platelet activity, and contractile strength, is more specifically useful in determining viability of stored blood products. Determining forces of contractility is particularly useful in diagnosis or prognosis of various diseases in patients. Because each of the components associated with clotting is the result of a myriad of intermediate steps, clot retraction is an excellent candidate for "funnel detection" where with one simple measurement it is possible to unmask a vast array of pathological stages. Funnel detection methods are particularly important in population screening studies.
  • Figure 1 shows Platelet interaction with surface immobilized collagen type I.
  • PJCM images show platelets in a flow field. On a gray scale, black indicates a distance from the surface of 4-12 nm; white of > 20-30 nm. The time after initiation of the experiment is shown at the right side of each panel. For this experiment the platelet count was reduced to 10,000 platelets per ⁇ l.
  • Figure 2 shows the evolution of an isolated thrombus at 100s ⁇
  • FIG. 2 shows the evolution of an isolated thrombus at 100s ⁇
  • Figure 4 is a photograph of the sterolithography model. The actual model was built to a scale of 300:1 and has a volume of 3.44 cm 3 .
  • FIG. 5 is a diagrammatic representation of two alternative designs for the retractometer of this invention.
  • Top panel (A) is a design in which the individual retractometers are connected through a system of communicating vessels sharing a common pressure transducer. This design allows the simultaneous measurement of several samples.
  • Bottom panel (B) is an alternate design having a clay plug closing the air filled capillary tube, which the operator "snaps" by bending it around the etching before starting the reading. The fluid inside the capillary then reaches the "zero level” corresponding to the hydrostatic pressure of the system. This design allows the direct measurement of the pressure without the need of electronics.
  • Figure 6 is a diagram showing the contracting element.
  • the upper panel of the figure represents the fibrin network before platelet contraction.
  • Figure 7 diagrammatically represents the origin of the forces developed by platelet contraction within a retractometer of this invention.
  • tension develops along the surface of the element, due to the "pull" between the contracting elements.
  • Figure 8 is a force analysis of the retractometer.
  • A is a diametrical cross-section in a plane passing through the center of the sphere.
  • B is a geometrical representation of two arbitrary but symmetrical vectors acting on the unit represented in A.
  • Figure 9 shows the mechanical model comprising a cantilever beam clamped at one end, subject to a constant bending moment.
  • Figure 11 Top is a photograph of one embodiment of an immersion mold. The prototype shown here was manufactured from stainless steel. Bottom: is a prototype of the flexible membrane of this invention.
  • Figure 12 is a graphic representation of a method used to pressure condition the membrane.
  • Figure 13 depicts the results of a preliminary experiment to show the feasibility of the proposed methodology.
  • the flexible membrane used was fabricated with latex with a thickness of 150 ⁇ m.
  • Citrated blood 11 ⁇ M Sodium Citrate was used.
  • Figure 14 illustrates the change in shape of the clots when separated from the membrane and cut open.
  • the photograph shows a petri dish with three sections of the clot.
  • a digitally enhanced magnification of the three samples shown is presented for better appreciation of the process.
  • Figure 15 is a schematic of the geometry of the cylindrical clot during contraction.
  • the contraction of the clot is considered to be isotropic (17).
  • the force F is measured directly by the force transducer.
  • the area A can be directly calculated from the measurement of the clot diameter.
  • the stress (F) can be estimated from this simple model.
  • Figure 16 is a comparison of the two methods used in this description. Both experiments were performed using platelet rich plasma.
  • Figure 17 is a schematic diagram of an electronic solenoid valve controller useful in simultaneous processing of many samples.
  • Figure 18 is a schematic representation of the fully automated system apparatus that can greatly increase the speed and ease of measuring platelet contractility in a number of retractometers, each having the same or different sample. Such a system is highly useful in screening populations and effectiveness of various drugs.
  • the present invention is based on the rationale that the development of a reliable method for the study of clot retraction will bring not only a useful tool to elucidate the mechanisms involved in the physiological mechanisms, but also an important tool for the monitoring of the overall well-being of the platelets in a blood sample.
  • This invention will potentially yield an important diagnostic tool for the monitoring and detection of pathological states, as well as an easy-to-use tool for the monitoring of platelet viability for transfusion purposes.
  • FIG. 1 The top panel in Figure 1 (0 minutes) shows the native spheroidal shape of the platelets, before activation. A few seconds after the initial platelet adhesion occurs, the first signs of activation are seen as dramatic shape changes and subsequent adhesion of the platelet membrane to the reactive surface. The other panels (2 and 4 minutes) show the activation and spreading that two single platelets undergo. Of notice is the large area that a single platelet can cover. This experiment shows the large amount of "membrane reservoir" contained by the relatively small platelets. After activation, the platelets initiate contraction, resulting in their deformation and clot formation. This phenomenon is not observed in the photographs shown here because the platelets are attached to a non- deformable surface (glass); however, the platelets increase the tension on their membrane due to the abovementioned cytoskeleton rearrangement.
  • Inventors describe their technical solution to create an upscale three- dimensional (3-D) model of a thrombus based on the information obtained with confocal microscopy.
  • a commonly used technique is stereolithography, which uses step-wise planar buildup of the object, based on the solidification of a photoresin by a laser beam. After each layer is cured, the object is lowered into a fluid resin pool by a distance equal to the vertical resolution of the system. This technique appears to be adequate in view of the complex geometries that it can handle.
  • the confocal microscopy images obtained were preprocessed with a 3x3 median filter, in order to minimize noise originated by the flow.
  • the images were then translated into a voxel space with a software package written by Inventors specifically for this purpose. This package maintains the relative positions of the measured objects in a cartesian three-dimensional space.
  • the voxel representation of the microscopic field was then used with AVS release 5.3 (Application Visual Systems, Inc., Waltham, MA) to render the three-dimensional field.
  • This software package allows the operator to interpolate a surface (isosurface) between contiguous voxels with an intensity above a preset threshold.
  • This isosurface is comprised of a series of triangles, each triangle being a "surface unit.”
  • the coordinates of these surface units were then stored in a geometry information file.
  • the geometry file was then translated into an "STL" file (standard input format for stereolithography) with a software package written by Inventors specifically for this purpose.
  • the STL file contains the coordinates of the vortices of the surface units and a normal vector pointing outside of the body of the object to be materialized. During the fabrication process, a scale factor was included to yield the desired dimensions.
  • a graphic representation of the file generation is shown in Figure 3.
  • the wall shear rate was 100 s_1 and the data presented correspond to images taken after 10 minutes of flow.
  • the geometry reconstructed here corresponds to the thrombus shown in the early time of Figure 2.
  • the upper left panel shows a summation of all the confocal images obtained from a real thrombus.
  • the upper right panel shows the topographical representation of the thrombus in pseudocolor.
  • the bar on the right shows the color code for the height in micrometers.
  • the lower left panel shows the 3-D representation rendered with AVS, as described above.
  • the lower right panel shows a graphic representation of the STL file.
  • the graph shows a wire model of the file, with the orientation identical to the previous panels.
  • the "hedgehog" appearance is due to normal vectors pointing outside of the body, as described in the method. For clarity, the normal vectors are shown in blue, and the wire model in white.
  • This type of graphic representation is not necessary for the actual fabrication process, but it is useful for error detection in the creation of the files and overall quality evaluation of the process. In the graph, the complexity of the surface and the large number of triangles necessary to reconstruct such a complex geometry can be appreciated.
  • Figure 4 shows the actual model described above built to a scale of 300:1 and has a volume of 3.44 cm .
  • the running time for this sample was 2.3 hours, using a FDM 2000 stereolithography machine (Stratasys, Ontario CA).
  • the orientation of this photography is similar but not identical to the orientation shown in Figures 2 and 3.
  • the performance of the device of this invention is based on the Laplace principle, in which the tension developed by the contraction of a sheet of platelets during clot retraction is transformed into an increase in pressure inside a semispherical flexible membrane.
  • Top panel (A) is a design in which the individual retractometers can be connected through a system of communicating vessels sharing a common pressure transducer. This design allows the simultaneous measurement of several samples.
  • a blood sample is placed inside (2) the rigid reservoir (3).
  • the thickness of the sample at 2 is A thin layer of mineral oil (light white oil, Sigma) is placed on top of the blood sample to avoid evaporation.
  • mineral oil light white oil, Sigma
  • Bottom panel (B) is a design in which the operator "snaps" the clay plug (7) of the air filled capillary tube, by bending it around the etching (scoring) (6) before starting the reading. Then the fluid inside the capillary reaches the "zero level” corresponding to the hydrostatic pressure of the system (details of the filling of the flexible membrane are given hereinbelow).
  • the filling fluid of the flexible membrane may contain a coloring agent for easier visualization.
  • the presence of the plug (7) prevents both fluid evaporation and changes in the fluid level by manipulation of the retractometer. This design allows the direct measurement of the pressure without the need of electronics.
  • Figure 6 is a diagrammatic representation of the contracting element described in the body of the text.
  • the upper object of Figure 6 represents the fibrin network before platelet contraction.
  • Figure 6 shows an isotropic retraction with a longitudinal strain of -0.5.
  • the bottom object shows the result of the isotropic contraction.
  • Figure 7 is a diagram representing the origin of the forces developed by platelet contraction in a retractometer.
  • tension develops along the surface of the element, due to the "pull" between the contracting elements.
  • the outer surface of the contracting element is free, the contraction of the element along the thickness h results in a decrease in volume and not a modification of tension on the contracting element.
  • the diagram on the right shows a free body taken from the contracting element.
  • a change in radius of the cylindrical component shown results in an increase in tension along the surface as shown, while a change in height (h) does not modify the tension along the surface of the plate.
  • Figure 8 represents a force analysis of the retractometer of this invention.
  • the flexible membrane of the retractometer is modeled for this analysis as a perfect sphere.
  • A is a diametrical cross-section in a plane passing through the center of the sphere as shown.
  • the resultant inner pressure Pi is homogeneously distributed on the inner surface of the retractometer.
  • the origin of all the vectors is the center of the sphere and all of them have the same magnitude.
  • the dotted line in this panel shows the arbitrary cross-section where the analysis is performed.
  • B is a geometrical representation of two arbitrary but symmetrical vectors acting on the unit represented in A.
  • the average tensile force can be easily calculated based on the measurement of the hydrostatic pressure inside the compartment defined by the flexible membrane. This analysis is exact for a perfect sphere. Although the retractometer deviates from this uniform stress field in the area where the flexible membrane attaches to the capillary tube or connecting tube, the stress analysis is an excellent approximation at the operational level and it is valid for the purposes of the design presented in this description.
  • Inventors decided to implement a system described by others (17), in which a cylindrical clot is immersed in ice-cold buffer to prevent platelet contraction. The clots are then anchored and held vertically to the bottom of the container at their lower end and to a force transducer to the upper end of the clot.
  • An isotonic force transducer was implemented.
  • the system is based on the single supported beam principle.
  • the mechanical model is a cantilever beam clamped at one end, subject to a constant bending moment. According to the following description:
  • M is the bending moment imposed by the load on the cantilevered beam
  • E is Young's modulus
  • I is a property of the cross-sectional geometry of the beam
  • the term on the left is the deflection of the beam (assuming a deflection much smaller than the length of the beam).
  • the deflection y( ⁇ ) can be calculated by using:
  • S is the transverse shear.
  • the bending moment and the transverse shear are related to the lateral load by:
  • the deflection is a linear function of the moment.
  • the first step in manufacturing of the flexible membrane is the fabrication of a suitable immersion mold.
  • Inventors fabricated a prototype mold from stainless steel.
  • the top panel is a photograph of the stainless steel immersion mold.
  • the ball has a diameter of 9/16" and the rod has a diameter of 3/32", but these could be of any suitable dimension.
  • the bottom panel shows an embodiment of the flexible membrane. This prototype was made to show the feasibility of fabrication using the immersion mold shown on top. This prototype embodiment was fabricated using urethane, but any other suitable material could be used. Due to the transparency of the material it is easy to study the thickness of the membrane.
  • the membranes would preferably be fabricated by experts in dip molding technology, for example by ACC Automation (Akron, OH). For this application, their 4-axis dipping system would be particularly suitable. Briefly, the system has 4 axis of operation (vertical, horizontal/pallet rotate and form spin), allowing the membrane coating to be uniform along the surface of the mold.
  • the equipment has a vertical stroke of 30 inches, a vertical axis speed range of 0.01-12 inch/sec, with 0.001 inch/sec speed increments.
  • Rotate axis positional range is 1440 degrees in 1 degree positional increments.
  • Rotate axis speed range is 0.1-60 degrees/sec in 0.1 degree speed increments.
  • Spin speed range is 10-100 RPM in 1 RPM increments.
  • Horizontal axis position is 18 inches in 0.01 inch positional increments.
  • Horizontal axis speed range is 0.01-4.0 inches /sec in 0.001 inches /sec/ Maximum payload capacity of 10 pounds.
  • the membranes are dried in an integrated force air convection electric oven, with programmable temperature control up to 200 °C.
  • the forms are spun in the oven to increase drying uniformity.
  • the membranes are fabricated using two coats of latex without thickening agent, in a similar fashion to fabricating condoms. Uniformity in thickness and mechanical properties of the membranes are highly reproducible.
  • the second step is to develop a suitable method for the filling of the membranes.
  • Inventors implemented a simple technique shown in Figure 12.
  • the membranes are coated with a suitable adhesive, for example, with a bovine collagen type I suspension as described elsewhere (2.5 mg/ml in 0.1 M acetic acid) (20). This method gave a firm adhesion of the clot onto latex membranes. It is expected that membranes of different materials may require other adhesives.
  • Membranes are pressure-conditioned as shown in Figure 12.
  • the flexible membrane is mounted on a sealing rubber stop with a needle inserted through it
  • the needle is connected to a two-way stop-cock, which in turn is connected to a syringe and another needle.
  • the reach of both needles is the same.
  • the syringe is used to slightly pressurize the flexible membrane.
  • access to the syringe is closed and the two needles are allowed to equilibrate the inner membrane pressure and the ambient pressure by siphoning the fluids.
  • This method is reliable in giving zero pressure readings with the use of a pressure transducer (Validyne DP 15-22, controlled by a Validyne CD379) immediately after inflation.
  • the third step is the adjusting of the fluid level inside the capillary at "zero pressure" level. As seen in Figure 5B, it would be desirable to control the level of the column inside the capillary to make the reading easier.
  • the height of the column in a capillary tube is dictated by the expression:
  • h is the height of the column
  • ⁇ is the surface tension of the fluid
  • p is the density of the fluid
  • r is the radius of the capillary tube
  • ⁇ is the wetting angle.
  • the glass capillaries are immersed in a 1% (w/w) NaOH water solution.
  • the container is heated to near boiling (bubble formation starting) (approx 90°C) and incubated for 10 minutes.
  • the solution is removed, and the capillaries are allowed to cool to room temperature.
  • the capillaries are then immersed in a 30% (w/w) H 2 O 2 solution, and heated to near boiling (approx 90°C) for one hour, 5 washed five times with deionized, deminerahzed water, tap dried and placed in a drying oven (250°C) for 12 hours.
  • Column heights were improved from 16 mm (with a 0.75 radius, untreated capillary) to 60 mm (with a 0.5 mm radius, hydrophilic modified capillary).
  • the fourth step is the calibration of the retractometer and comparison of the l o results to an alternative, known method.
  • Figure 13 demonstrate the feasibility and validity of the principle of operation of the methodology.
  • the stresses along the thickness of the clot are not uniform, due to the geometry of the retractometer. This lack of uniformity in stresses must, therefore, result in "shearing strain" across the thickness of the clot.
  • This is seen macroscopically in Figure 14 as twisting of the clots.
  • Figure 14 shows a petri dish with three sections of the clot. A digitally enhanced magnification of the three samples shown is presented for better appreciation of the process. It should be noted that the larger the deformation the larger is the residual stress. The large twisting deformation is due to the non-uniform increase in tension along the thickness of the wall of the clots.
  • Cylindrical clots are obtained by pouring a human platelet-rich plasma
  • Figure 15 outlines schematically the geometry of the cylindrical clot during contraction.
  • the contraction of the clot is considered to be isotropic (17).
  • the force F is directly measured by the force transducer.
  • the area A can be directly calculated from the measurement of the clot diameter.
  • the stress (F) can be estimated from this simple model.
  • the electronic solenoid valve controller circuit is shown in Figure 17.
  • a 10 x 16 array was implemented.
  • the circuit labeled digital row selection is meant for an alternative computer control.
  • a voltage is generated at the output of the circuit, which is used to select the row on the right hand circuits.
  • the voltage used for row selection can also be selected manually via a potentiometer part of a voltage divider.
  • Implementation of these circuits allows the operator to select the row either manually or via digital input.
  • the operational amplifier (Row Gain) is intended to give the maximal gain of the voltage divider for row selection. The use of this is to select manually the maximal row number to be read in a given cycle.
  • This non-inverting input of the operation amplifier (op amp) is amplified and sent to an A/D converter, implemented by the comparators and the priority encoder. Should the control be exclusively digital, this part of the circuit is obsolete, in which case the already digital input should be sent directly to the 1 of 10 decoder (10 is arbitrarily chosen in this case, and the total number of rows can be different). The final result of this architecture is that only one row is activated at a time.
  • Inventors chose to add a timer assuming that the column selection is done in a continuous sweep.
  • the timer shown in the lower left corner of the diagram ( Figure 17) has a feedback loop controlled by a potentiometer that allows the operator to control the sweeping rate.
  • the output of the timer serves as the input for a 4-bit counter, the output of the counter is input into a 1 of 16 decoder to select only one column at a time.
  • FIG. 18 Another embodiment of an electronic solenoid valve controller of this invention is shown in Figure 18.
  • an 8051 microprocessor is used.
  • Three pins of the microprocessor actuate a syringe pump. Pin one is used to rum the pump on and the other two to move the pump piston either up or down.
  • the pump is directly connected to the hydraulics (retractometers) of the system.
  • the motor of the pump is connected to a voltage divider that yields a voltage used to establish the position of the piston of the pump. This readout position voltage is entered through an analog to digital (A/D) converter to the microprocessor.
  • Other pins of the microprocessor are connected to each one of the solenoid valves used in the array.
  • a series of eight valves are used to measure each one of the retractometer samples and two others are used to provide protection to the system.
  • One of the protection valves is located at the entrance of the pressure transducer, and its role is to prevent damage to the system due to the operation of the pump.
  • the other valve is located to provide access to a fluid reservoir. This valve is used in this example to fill the syringe prior to the beginning of the experiments.
  • the output voltage of the pressure transducer is entered into the A D converter and subsequently to the microprocessor.
  • the subroutines were burnt into the microprocessor.
  • the microprocessor reads all initial pressures of all the samples by opening each individual sample valve, followed by opening of the protection valve.
  • the voltage from the pressure transducer is measured and stored in the temporary memory of the microprocessor. This process is repeated until all the initial pressure values are registered.
  • the previous pressure value for the valve that will be measured is taken as the target value. Then the value of the hydraulics is taken, having only the protection valve opened. The pump is then actuated (either up or down depending on the relative value of the target pressure) until the target pressure value is reached. The sample valve is then opened, the pressure is measured, and the sample valve is closed. The measured values are sent to a text file in a PC computer via a serial port. The new measured value for each valve becomes the next target value. These cycles are repeated until the end of the experiment.
  • Rho a connection between membrane receptor signalling and the cytoskeleton. Trends Cell Biol 3:14-310.

Abstract

La présente invention se rapporte à un appareil et une méthode de mesure de la contractilité des plaquettes, ledit appareil étant appelé 'rétractomètre''; ainsi qu'un dispositif et une méthode de mesure automatique de la contractilité des plaquettes dans une pluralité d'échantillons, au moyen d'un réseau d'unités de rétractomètre et d'un dispositif de commande électronique de vanne électromagnétique afin d'effectuer le criblage de manière entièrement automatique dans des situations cliniques et de recherche et afin de réduire les coûts en termes de main-d'oeuvre.
PCT/US2003/024052 2003-01-08 2003-08-01 Appareil et methode de mesure de la contractilite des plaquettes WO2004062570A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9140684B2 (en) 2011-10-27 2015-09-22 University Of Washington Through Its Center For Commercialization Device to expose cells to fluid shear forces and associated systems and methods

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007084544A2 (fr) * 2006-01-17 2007-07-26 Regenerx Biopharmaceuticals, Inc. Procedes de traitement ou de prevention d’endommagement de tissus causes par une augmentation du debit sanguin
US7976795B2 (en) * 2006-01-19 2011-07-12 Rheonix, Inc. Microfluidic systems
US8609039B2 (en) * 2006-01-19 2013-12-17 Rheonix, Inc. Microfluidic systems and control methods
US20090287076A1 (en) * 2007-12-18 2009-11-19 Boyden Edward S System, devices, and methods for detecting occlusions in a biological subject
US20090292222A1 (en) * 2008-05-14 2009-11-26 Searete Llc Circulatory monitoring systems and methods
US9717896B2 (en) * 2007-12-18 2017-08-01 Gearbox, Llc Treatment indications informed by a priori implant information
US9672471B2 (en) * 2007-12-18 2017-06-06 Gearbox Llc Systems, devices, and methods for detecting occlusions in a biological subject including spectral learning
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US20100036209A1 (en) * 2008-08-07 2010-02-11 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Circulatory monitoring systems and methods
US20090163856A1 (en) * 2007-12-19 2009-06-25 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Treatment indications informed by a prior implant information
US20100036263A1 (en) * 2008-08-07 2010-02-11 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Circulatory monitoring systems and methods
US20090292213A1 (en) * 2008-05-21 2009-11-26 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Circulatory monitoring systems and methods
US8280484B2 (en) * 2007-12-18 2012-10-02 The Invention Science Fund I, Llc System, devices, and methods for detecting occlusions in a biological subject
US20090287093A1 (en) * 2008-05-15 2009-11-19 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Circulatory monitoring systems and methods
US20090281412A1 (en) * 2007-12-18 2009-11-12 Searete Llc, A Limited Liability Corporation Of The State Of Delaware System, devices, and methods for detecting occlusions in a biological subject
US8636670B2 (en) * 2008-05-13 2014-01-28 The Invention Science Fund I, Llc Circulatory monitoring systems and methods
US20090287120A1 (en) * 2007-12-18 2009-11-19 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Circulatory monitoring systems and methods
US20090287110A1 (en) * 2008-05-14 2009-11-19 Searete Llc Circulatory monitoring systems and methods
EP3427034A4 (fr) * 2016-03-07 2019-12-18 The Regents of The University of Michigan Plateforme de test de coagulation de sang total à l'échelle microscopique

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4986964A (en) * 1990-04-19 1991-01-22 Center For Innovative Technology Clot retractometer
US5205159A (en) * 1992-01-17 1993-04-27 Virginia Commonwealth University Apparatus and method for measuring clot elastic modulus and force development on the same blood sample
US5293772A (en) * 1992-01-17 1994-03-15 Center For Innovative Technology Instrumentation and method for evaluating platelet performance during clotting and dissolution of blood clots and for evaluating erythrocyte flexibility
US5441892A (en) * 1992-10-15 1995-08-15 Medtronic Hemptec, Inc. Blood clot mass measuring technique
US5854423A (en) * 1996-03-20 1998-12-29 Venegas; Jose G. Apparatus and method for assessment of visco-elasticity and shear adherence strength properties of blood clots
US5925569A (en) * 1986-09-10 1999-07-20 Xylum Corporation Blood testing device and method
US6159741A (en) * 1996-04-30 2000-12-12 Dr. Michael Kratzer Gmbh Process for measuring blood platelet aggregation or blood coagulation
US6573104B2 (en) * 2001-05-10 2003-06-03 Hemodyne, Incorporated Disposable cup and cone used in blood analysis instrumentation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5455009A (en) * 1993-09-14 1995-10-03 Becton, Dickinson And Company Blood collection assembly including clot-accelerating plastic insert
US5656499A (en) * 1994-08-01 1997-08-12 Abbott Laboratories Method for performing automated hematology and cytometry analysis
US6613573B1 (en) * 1999-02-22 2003-09-02 Haemoscope Corporation Method and apparatus for monitoring anti-platelet agents

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925569A (en) * 1986-09-10 1999-07-20 Xylum Corporation Blood testing device and method
US4986964A (en) * 1990-04-19 1991-01-22 Center For Innovative Technology Clot retractometer
US5205159A (en) * 1992-01-17 1993-04-27 Virginia Commonwealth University Apparatus and method for measuring clot elastic modulus and force development on the same blood sample
US5293772A (en) * 1992-01-17 1994-03-15 Center For Innovative Technology Instrumentation and method for evaluating platelet performance during clotting and dissolution of blood clots and for evaluating erythrocyte flexibility
US5441892A (en) * 1992-10-15 1995-08-15 Medtronic Hemptec, Inc. Blood clot mass measuring technique
US5854423A (en) * 1996-03-20 1998-12-29 Venegas; Jose G. Apparatus and method for assessment of visco-elasticity and shear adherence strength properties of blood clots
US6159741A (en) * 1996-04-30 2000-12-12 Dr. Michael Kratzer Gmbh Process for measuring blood platelet aggregation or blood coagulation
US6573104B2 (en) * 2001-05-10 2003-06-03 Hemodyne, Incorporated Disposable cup and cone used in blood analysis instrumentation

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9140684B2 (en) 2011-10-27 2015-09-22 University Of Washington Through Its Center For Commercialization Device to expose cells to fluid shear forces and associated systems and methods
US9213024B2 (en) 2011-10-27 2015-12-15 University Of Washington Microfluidic devices for measuring platelet coagulation and associated systems and methods
US10006900B2 (en) 2011-10-27 2018-06-26 University Of Washington Devices to expose cells to fluid shear forces and associated systems and methods

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US20090130744A1 (en) 2009-05-21
WO2004062570A3 (fr) 2005-07-07
US20050027184A1 (en) 2005-02-03
AU2003257983A8 (en) 2004-08-10
US20080286823A1 (en) 2008-11-20
US7807106B2 (en) 2010-10-05
EP1618373A2 (fr) 2006-01-25
US7335335B2 (en) 2008-02-26

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